An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition

Extracellular matrix (ECM) is a dynamic network of proteins, proteoglycans and glycosaminoglycans, providing structure to the tissue and biochemical and biomechanical instructions to the resident cells. In fibrosis, the composition and the organization of the ECM are altered, and these changes influence cellular behaviour. Biochemical (i. e. protein composition) and biomechanical changes in ECM take place simultaneously in vivo. Investigating these changes individually in vitro to examine their (patho)physiological effects has been difficult. In this study, we generated an in vitro model to reflect the altered mechanics of a fibrotic microenvironment through applying fibre crosslinking via ruthenium/sodium persulfate crosslinking on native lung ECM-derived hydrogels. Crosslinking of the hydrogels without changing the biochemical composition of the ECM resulted in increased stiffness and decreased viscoelastic stress relaxation. The altered stress relaxation behaviour was explained using a generalized Maxwell model. Fibre analysis of the hydrogels showed that crosslinked hydrogels had a higher percentage of matrix with a high density and a shorter average fibre length. Fibroblasts seeded on ruthenium-crosslinked lung ECM-derived hydrogels showed myofibroblastic differentiation with a loss of spindle-like morphology together with greater α-smooth muscle actin (α-SMA) expression, increased nuclear area and circularity without any decrease in the viability, compared with the fibroblasts seeded on the native lung-derived ECM hydrogels. In summary, ruthenium crosslinking of native ECM-derived hydrogels provides an exciting opportunity to alter the biomechanical properties of the ECM-derived hydrogels while maintaining the protein composition of the ECM to study the influence of mechanics during fibrotic lung diseases. STATEMENT OF SIGNIFICANCE: Fibrotic lung disease is characterized by changes in composition and excessive deposition of extracellular matrix (ECM). ECM fibre structure also changes due to crosslinking, which results in mechanical changes. Separating the changes in composition and mechanical properties has been difficult to date. In this study, we developed an in vitro model that allows alteration of the mechanical changes alone by applying fibre crosslinking in native lung ECM-derived hydrogels. Characterisations of the crosslinked hydrogels indicated the model mimicked mechanical properties of fibrotic lung tissue and reflected altered fibre organisation. This ECM-based fibrosis model provides a method to preserve the native protein composition while altering the mechanical properties providing an important tool not only for lung but also other organ fibrosis

Architecture and Composition Dictate Viscoelastic Properties of Organ-Derived Extracellular Matrix Hydrogels

The proteins and polysaccharides of the extracellular matrix (ECM) provide architectural support as well as biochemical and biophysical instruction to cells. Decellularized, ECM hydrogels replicate in vivo functions. The ECM’s elasticity and water retention renders it viscoelastic. In this study, we compared the viscoelastic properties of ECM hydrogels derived from the skin, lung and (cardiac) left ventricle and mathematically modelled these data with a generalized Maxwell model. ECM hydrogels from the skin, lung and cardiac left ventricle (LV) were subjected to a stress relaxation test under uniaxial low-load compression at a 20%/s strain rate and the viscoelasticity determined. Stress relaxation data were modelled according to Maxwell. Physical data were compared with protein and sulfated GAGs composition and ultrastructure SEM. We show that the skin-ECM relaxed faster and had a lower elastic modulus than the lung-ECM and the LV-ECM. The skin-ECM had two Maxwell elements, the lung-ECM and the LV-ECM had three. The skin-ECM had a higher number of sulfated GAGs, and a highly porous surface, while both the LV-ECM and the lung-ECM had homogenous surfaces with localized porous regions. Our results show that the elasticity of ECM hydrogels, but also their viscoelastic relaxation and gelling behavior, was organ dependent. Part of these physical features correlated with their biochemical composition and ultrastructure

Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed

Higher Chain Length Distribution in Debranched Type-3 Resistant Starches (RS3) Increases TLR Signaling and Supports Dendritic Cell Cytokine Production

Scope: Resistant starches (RSs) are classically considered to elicit health benefits through fermentation. However, it is recently shown that RSs can also support health by direct immune interactions. Therefore, it has been hypothesized that the structural traits of RSs might impact the health benefits associated with their consumption. Methods and results: Effects of crystallinity, molecular weight, and chain length distribution of RSs are determined on immune Toll-like receptors (TLRs), dendritic cells (DCs), and T-cell cytokines production. To this end, four type-3 RSs (RS3) are compared, namely Paselli WFR, JD150, debranched Etenia, and Amylose fraction V, which are extracted from potatoes and enzymatically modified. Dextrose equivalent seems to be the most important feature influencing immune signaling via activation of TLRs. TLR2 and TLR4 are most strongly stimulated. Especially Paselli WFR is a potent activator of multiple receptors. Moreover, the presence of amylose, even to residual levels, enhances DC and T-cell cytokine responses. Paselli WFR and Amylose fraction V influence T-cell polarization. Conclusions: It has been shown here that chain length and particularly dextrose equivalent are critical features for immune activation. This knowledge might lead to tailoring and design of immune-active RS formulations.</p

Higher Chain Length Distribution in Debranched Type-3 Resistant Starches (RS3) Increases TLR Signaling and Supports Dendritic Cell Cytokine Production

Scope Resistant starches (RSs) are classically considered to elicit health benefits through fermentation. However, it is recently shown that RSs can also support health by direct immune interactions. Therefore, it has been hypothesized that the structural traits of RSs might impact the health benefits associated with their consumption. Methods and results Effects of crystallinity, molecular weight, and chain length distribution of RSs are determined on immune Toll-like receptors (TLRs), dendritic cells (DCs), and T-cell cytokines production. To this end, four type-3 RSs (RS3) are compared, namely Paselli WFR, JD150, debranched Etenia, and Amylose fraction V, which are extracted from potatoes and enzymatically modified. Dextrose equivalent seems to be the most important feature influencing immune signaling via activation of TLRs. TLR2 and TLR4 are most strongly stimulated. Especially Paselli WFR is a potent activator of multiple receptors. Moreover, the presence of amylose, even to residual levels, enhances DC and T-cell cytokine responses. Paselli WFR and Amylose fraction V influence T-cell polarization. Conclusions It has been shown here that chain length and particularly dextrose equivalent are critical features for immune activation. This knowledge might lead to tailoring and design of immune-active RS formulations